Cilia and flagella are evolutionarily conserved organelles with important biological roles in motility, sensation and signaling. They are essential in the life cycles of most eukaryotic organisms, and cilia are found in nearly every human cell. The goal of this research is to determine how regulatory signals change the activity of the molecular motor protein dynein, and how dynein activity is regulated in motile cilia and flagella to generate proper beating. Our laboratory opened a new window into the three-dimensional organization and protein composition of cilia and flagella, and recently discovered previously unseen structures that regulate dynein function and/or are essential for flagellar assembly. Building on a strong base of preliminary data gathered in the preceding project period, this proposal directly addresses key open questions in the field in three specific aims that are directed at understanding the three-dimensional structure, subunit composition, protein interactions and the regulatory functions of the following three major regulatory and signal transduction complexes in cilia and flagella: (1) the nexin-dynein regulatory complex, (2) the I1 inner dynein complex, and (3) radial spoke 3 in organisms with radial spoke triplets. The project uses a multi-disciplinary approach and cutting-edge techniques, combining in situ molecular imaging by cryo-electron tomography and image processing, biochemical and mass spectrometric analyses, integrated structural-genetics approaches and protein labeling techniques to directly visualize gene products in cells. The proposed research will contribute fundamental knowledge and a deeper understanding of the mechanisms underlying motor protein function and control on a molecular level and of the functional organization of cilia and flagella.